The general goal of this project, which started in 1992, was to investigate the energetic bases of all levels of protein structure, including specific protein complexes. In previous years we studied the factors responsible for formation of the tertiary structure of globular proteins. For the next five-years we propose to concentrate on the next level of molecular organization, namely protein complexes with DNA. The problem of protein interaction with DNA is now attracting increasing attention and is intensively studied by structural methods. The main difference between the intramolecular process of protein folding and intermolecular formation of macromolecular complexes is that the later involve much fewer interactions and are more affected by the environmental conditions. Correspondingly, the energies of formation of the complexes are smaller and their measurement under varying conditions and the thermodynamic analysis require more sophisticated experimental methods. Using supersensitive scanning calorimetry, we have found that the heat capacity of complexes depends significantly on temperature, i.e. upon heating, energy accumulates gradually in thermal fluctuations of their structure that increase to some critical level, whereupon the structure bursts, i.e. undergoes cooperative dissociation/unfolding. Thus the energetics of macromolecular complexes depends significantly on the thermal, dynamic properties of these complexes and their components. Correspondingly, solution of this problem requires simultaneous studies of (a) the thermodynamic characteristics of association of the components into a complex (the enthalpy and entropy of association) at different temperatures and solvent conditions by isothermal titration calorimetry (ITC), (b) study of the thermal properties (the heat capacity) of the complex and all its components, over a broad temperature range and solvent conditions by differential scanning calorimetry (DSC), and (c) the study of the dynamic properties (mobility of groups) in this temperature range and solvent conditions by NMR. This project proposes to realize these objectives with combined investigations on several extended groups of DNA-binding proteins and their complexes.